Self-assembling polynucleotide delivery system

ABSTRACT

This invention provides a self-assembling polynucleotide delivery system comprising components aiding in the delivery of the polynucleotide to the desired address which are associated via noncovalent interactions with the polynucleotide. The components of this system include DNA-masking components, cell recognition components, charge-neutralization and membrane-permeabilization components, and subcellular localization components. Specific compounds useful in this system are also provided.

CROSS-REFERENCE TO RELATED CASE

This is a continuation of appplication Ser. No. 07/913,669 filed on Jul.14, 1992, entitled SELF-ASSEMBLING POLYNUCLEOTIDE DELIVERY SYSTEM nowabandoned which is a continuation-in-part of U.S. Ser. No. 07/864,876,filed Apr. 3, 1992, now abandoned.

TECHNICAL FIELD

This invention is in the field of oligonucleotide delivery and genetherapy. In particular this invention is directed to a self-assemblingpolynucleotide delivery system comprising components aiding in thedelivery of the polynucleotide to the desired address which areassociated via noncovalent interactions with the polynucleotide. Thecomponents of this system include DNA-masking components, cellrecognition components, charge-neutralization andmembrane-permeabilization components, and subcellular localizationcomponents.

BACKGROUND ART

Cystic Fibrosis (CF) is a fatal recessive genetic disease characterizedby abnormalities in chloride transport (McPherson & Dorner, 1991). Thelocus of the disease has been traced to mutations in the gene encodingthe cystic fibrosis transmembrane conductance regulator (CFTR). J. R.Riordan et al., Science (1989) 245:1066-1073; B. Kerem et al., Science(1989) 245:1073-1080. Correction of the underlying gene defect bycomplementation or replacement of the defective CFTR is the ultimatecure for CF. Gene therapy, the in vivo delivery and expression of genes,is a fast-developing science that can be used to replace defectivegenes.

Gene therapy is already feasible. T. Friedmann, Science (1989)244:1275-1281; M. Bluestone, Biotechnol (1992) 10:132-134. Systems andpolymers for delivery of polynucleotides are known in the art. P. L.Felgner, Adv Drug Delivery Rev (1990) 5:163-187. Adenoviral vectors havebeen used to transfer CFTR to the cotton rat lung in vivo. M. A.Rosenfeld et al., Cell (1992) 68:143-155. Although high levels oftransfection in vivo have been reported with the adenoviral vectors,non-viral delivery systems have a number of advantages and should bevigorously developed. Rosenfeld et al., supra; M. A. Rosenfeld et al.,Science (1991) 252:431-434.

During the past decade, a number of methods have been developed tointroduce functional genes into mammalian cells in vitro. Thesetechniques are applicable to gene therapy if the target cells can beremoved from the body, treated, and the transfected cells amplified andthen returned to the patient. This option is not possible for CFpatients. At present the best in vivo transfection efficiencies areobtained with retroviruses (Bluestone, supra) and adenoviruses(Rosenfeld et al., supra). However the efficiency is variable and aconcern is that virus based gene delivery might cause viral infection orcancer. Initial human clinical trials have revealed no acutecomplications of retroviral vectors but the possibility of long-termcomplications mandate careful patient monitoring. K. Cornetta et al.,Human Gene Ther (1991) 2:3-14.

The risks of using viral based vectors and the conceptual advantages inusing plasmid DNA constructs for gene therapy (discussed in P. L.Felgner et al., Nature (1991) 349:351-352) have led to a paralleldevelopment of various physical and chemical methods for gene transfer.The most intensely studied systems involve treatment of the cells withcalcium phosphate or a cationic facilitator (Felgner et al., supra).Other popular methods involve DNA injection during physical puncture ofthe membrane (M. R. Capecchi, Cell (1980) 22:479-485) or passive uptakeduring permeabilization or abrasion of the cellular membrane (Felgner etal., supra). Each method is intrinsically aggressive and applicable onlyin vitro.

This invention is in the field of direct gene delivery that does notinvolve the use of viral vehicles. A non-viral carrier for gene deliverymust be able to surmount many barriers: it must survive in circulation,it must be able to target the cell of choice, it must be able tointroduce DNA into the cytoplasm, and it must be able to transport theDNA into the nucleus.

Masking. One concern about the direct delivery of genes in vivo is theability of the polynucleotide to survive in circulation long enough toarrive at the desired cellular destination. “Masking”, or protecting thepolynucleotides is one way to address this concern.

Microparticulates (such as the erythrocyte ghost, reconstituted viralenvelopes and liposomes) have been used in part as protection in genetransfer. C. Nicolau et al., Crit Rev Ther Drug Carr Sys (1989)6:239-271; R. J. Mannio et al., Biotechniques (1988) 6:682-690. The mostsuccessful liposome system uses the cationic lipid reagentN-[1(-2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA).P. L. Felgner et al., Proc Natl Acad Sci (USA) (1987) 84:7413-7417.DOTMA is mixed with phosphatidylethanolamine (PE) to form the reagentLipofectin™. The advantage of using Lipofectin™ is that the cationicliposome is simply mixed with the DNA and added to the cell. It is notnecessary to encapsulate the DNA inside of the liposome with thecationic reagents. Lipofectin™ has been used to transfect reporter genesinto human lung epithelial cells in culture (L. Lu et al., Pflugers Arch(1989) 415:198-203), to introduce the CAT gene into rats byintratracheal route (T. A. Hazinski et al., Am J Respir Cell Mol Biol(1991) 4:206-209) and to introduce the CAT gene into mice by theintratracheal and intravenous route (K. L. Brigham et al., Am J Med Sci(1989) 298:278-281; A. Bout et al., “Abstracts of the 1991 CysticFibrosis Conference”, Abstract no. 87 (1991)). About 50% of the airwayepithelial cells transiently expressed the β galactosidase reporter gene(Hazinski et al., supra) but the level of expression was notquantitated. When chloramphenicol acetyltransferase (CAT) attached to asteroid sensitive promoter was transfected into rat lung, expressioncould be positively regulated by dexamethasone. Hazinski et al., supra.Cytotoxicity is a problem with high concentrations of Lipofectin™.

Substitutes for DOTMA include lipopolyamine (J. Loeffler et al., JNeurochem (1990) 54:1812-1815), lipophilic polylysines (X. Zhou et al.,Biochim Biophys Acta (1991) 1065:8-14) and a cationic cholesterol (X.Gao et al., Biochem Biophys Res Comm (1991) 179:280-285). These havebeen used to mediate gene transfer in culture. Although there is someimprovement over transfection rates observed with Lipofectin™ (aboutthreefold), toxicity remains a problem. Studies on the mechanismresponsible for transfection using the cationic lipids have been notablylacking. The past approach has been to synthesize different cationiclipids and try them in transfection assays, rather than tosystematically study how the delivery systems introduce DNA into thecell. DOTMA/PE liposomes can undergo bilayer fusion with anionicliposomes (N. Duzgunes et al., Biochem (1989) 28:9179-9184) whichsuggests that direct entry of the DNA via the plasma membrane isinvolved with DOTMA's mode of action. High efficiency transfection usingcationic lipids systems requires the inclusion of PE, possibly becausePE can form intramembrane lipid intermediates which facilitate membranefusion. The role of PE in membrane permeabilization and fusion has beenextensively studied. E.g., M.-Z. Lai et al., Biochem (1985)24:1646-1653; H. Ellens et al., Biochem (1986) 25:285-294; J. Bentz etal., Biochem (1987) 26:2105-2116).

Cellular Targeting. Efficient gene transfer requires targeting of theDNA to the cell of choice. Recently, procedures based upon receptormediated endocytosis have been described for gene transfer. G. Y. Wu etal., J Biol Chem (1987) 262:4429; G. Y. Wu et al., J Biol Chem (1988)263:14621-14624. A cell-specific ligand-polylysine complex is bound tonucleic acids through charge interactions. The resulting complex istaken up by the target cells. Wu et al., supra, reported efficienttransfection of the human hepatoma cell line HepG2 and of rathepatocytes in vivo using this delivery system with asialoorosomucoid asa ligand. Huckett et al., Biochem Pharmacol (1990) 40:253-263, reportedstable expression of an enzymatic activity in HepG2 cells followinginsulin-directed targeting. Finally Wagner et al., Proc Natl Acad Sci(USA) (1990) 87:3410-3414 and (1991) 88:4255-4259 observedtransferrin-polycation-mediated delivery of a plasmid into the humanleukemic cell line K-562 and subsequent expression of the encodedluciferase gene. However, the described delivery systems are based uponhigh molecular weight targeting proteins linked to DNA through apolylysine linker. These large ligand-polycation conjugates areheterogenous in size and composition, not chemically well-defined, anddifficult to prepare in a reproducible fashion (Wu et al., supra; Wagneret al., supra). Moreover, in many of the receptor mediated systems,chloroquine or other disruptors of intracellular trafficking arerequired for high levels of transfection. In one study, adenovirus hasbeen used to enhance gene delivery of the receptor mediated systems. D.T. Curiel et al., Proc Natl Acad Sci (USA) (1991) 88:8850-8854.

Together these studies show that genes can be delivered into theinterior of mammalian cells by receptor mediated endocytosis and afraction of the exogenous DNA escapes degradation, enters the nucleus,and is expressed. The level of expression is low, probably due to thelimited entry of the foreign DNA into the cytoplasm.

Charge Neutralization and Membrane Permeabilization. Direct delivery ofgenes is aided by the ability to neutralize the large negative charge onthe polynucleotide, and the (often concomitant) ability to permeabilizethe membrane of the targeted cell. The use of polycations to neutralizethe polynucleotide charge and aid in the membrane permeabilization andtranslocation is well known. Felgner, supra. Cationic lipids have alsobeen used for this purpose. P. L. Felgner et al., Proc Natl Acad Sci(USA) (1987) 84:7413-7417; U.S. Pat. No. 4,946,787 to Eppstein et al.Certain cationic lipids termed lipopolyamines and lipointercalants arealso known. J.-P. Behr, Tet Lett (1986) 27:5861-5864.

Subcellular Localization. Once the polynucleotide has entered thetargeted cell, direct delivery of genes would be aided by the ability todirect the genes to the proper subcellular location. One obvious targetfor the delivery of deoxyribonucleotides is the nucleus. Ligands knownto aid in this process are nuclear localization peptides, or proteinscontaining these nuclear localization sequences. C. Dingwall et al.,TIBS (1991) 16:478-481.

Y. Kaneda et al., Science (1989) 243:375-378, showed that thetransfection efficiency obtained with reconstituted viral envelopes isincreased when the foreign gene is co-delivered into the target cellswith nuclear proteins. DNA mixed with nuclear proteins exhibit a modestincrease in transfection over DNA that was mixed with albumin (Kaneda etal.). The assumption is that the DNA is incorporated into the nucleusmore readily when proteins containing the nuclear localization sequence(NLS) SEQ ID NO:1 (P. A. Silver, Cell (1991) 64:489-497) are associatedwith the plasmid. The NLS on a protein designates it for transportthrough the nuclear pore. Nuclear localization sequences of 14 aminoacids have been attached to a variety of macromolecules and even goldparticles (150 A diameter) and, when introduced into the cytoplasm, theyare rapidly incorporated into the nucleus (D. R. Findlay et al., J CellSci Supp (1989) 11:225-242; Silver, supra). The suggestion that nuclearentry is rate limiting for successful, stable transfection is alsosupported by the finding that plasmid DNA microinjected into thecytoplasm is unable to bring about transfection of cells (notransfection out of 1000 cytoplasmic injections, whereas microinjectionof plasmids directly into the nucleus results in transfection in greaterthan 50% of the microinjected cells. Cappechi, supra. If the attachmentof nuclear localization signals on the plasmid leads to transport ofplasmid DNA into the nucleus, the transfection efficiency shouldincrease. We propose a novel method to attach NLS and other ligands tothe desired polynucleotide.

Finally, investigators have demonstrated that transfection efficienciesincrease when DNA is condensed using various cationic proteins. T. I.Tikchonenko et al., Gene (1988) 63:321-330; M. Bottger et al., BiochimBiophys Acta (1988) 950:221-228; Wagner et al., supra. The reason whyDNA condensation increases transfection is not readily apparent, it mayincrease cellular uptake of DNA (Wagner et al., supra) but it also candecrease susceptibility of the DNA to nuclease activity which may resultin higher amounts of intact DNA in the cell.

Polynucleotide Association. Direct delivery of genes associated with oneof the above-discussed classes of molecules, is further aided by theability of those components to remain associated with the DNA. Wu etal., supra, associated their receptor ligand with the polynucleotide bycovalently attaching the ligand to the polycation polylysine. Wagner etal., Bioconjugate Chem. (1991) 2:226-231, in addition to polylysine,also covalently attached the ligand to a DNA intercalator, ethidiumhomodimer(5,5′-diazadecamethylenebis(3,8-diamino-6-phenylphenanthridium)dichloride dihydrochloride). P. E. Nielsen, Eur J Biochem (1982)122:283-289, associated photoaffinity labels to DNA by covalentattachment to 9-aminoacridine and certain bis-acridines.

None of the above references describe a system for polynucleotidedelivery aimed at multiple aspects of the problems involved in bringinga circulating polynucleotide to a targeted subcellular location of atargeted cell. This invention addresses those problems by associatingthe polynucleotide with a combination of one or more of the followingfunctional components: DNA-masking components, cell recognitioncomponents, charge-neutralization and membrane-permeabilizationcomponents, and subcellular localization components.

SUMMARY OF THE INVENTION

In light of the aforementioned problems of direct gene delivery, thisinvention contemplates a self-assembling polynucleotide delivery systemutilizing a combination of one or more, preferably two or more of thefollowing functional components: DNA-masking components, cellrecognition components, charge-neutralization andmembrane-permeabilization components, and subcellular localizationcomponents. Each component in this system is able to perform itsindicated function and also be capable of assembling or disassemblingwith the polynucleotide as required. For example, certain components mayhave to dissociate from the polynucleotide in order for it to performits desired function.

It is accordingly a primary object of this invention to provide acomposition for presenting a polynucleotide to a subcellular componentof a eukaryotic cell comprising the polynucleotide associated with amembrane-permeabilizing component capable of transporting thepolynucleotide across the cytoplasmic membrane of the eukaryotic cell.

It is another object of this invention to provide a composition forpresenting a polynucleotide to the nucleus of a eukaryotic cellcomprising the polynucleotide associated with a cell recognitioncomponent capable of recognizing the eukaryotic cell.

It is yet another object of this invention to provide a composition forpresenting a polynucleotide to the nucleus of a eukaryotic cellcomprising the polynucleotide associated with both a cell recognitioncomponent capable of recognizing the eukaryotic cell, and amembrane-permeabilizing component capable of transporting thepolynucleotide across the cytoplasmic membrane of the eukaryotic cell.

It is a further object of this invention to provide a composition forpresenting a polynucleotide to a subcellular component of a eukaryoticcell comprising the polynucleotide associated with asubcellular-localization component capable of delivering thepolynucleotide from the cytoplasm of the eukaryotic cell to asubcellular component of the eukaryotic cell.

It is still a further object of this invention to provide a compositionfor presenting a polynucleotide to a subcellular component of aeukaryotic cell comprising the polynucleotide, a cell recognitioncomponent capable of recognizing said eukaryotic cell, amembrane-permeabilizing component capable of transporting thepolynucleotide across the cytoplasmic membrane of said eukaryotic cell,a subcellular-localization component capable of delivering thepolynucleotide from the cytoplasm of said eukaryotic cell to asubcellular component of said eukaryotic cell, and a masking componentcapable of increasing the circulatory half-life of the polynucleotide.

It is another object of this invention to provide a component useful inself-assembling polynucleotide delivery systems having the formula

wherein each of n and m is independently an integer of 1 to 20, p is aninteger of 0 to 20, Ar₁ and Ar₂ are independently selected from thegroup consisting of ethidium bromide, acridine, mitoxantrone,oxazolopyridocarbazole, ellipticine and N-methyl-2,7-diazapyrenium, andderivatives thereof, X is a reactive coupling group, and Y is selectedfrom the group consisting of masking compound, cell surface receptorligands, subcellular localization sequences, and membrane permeabilizingcomponents.

It is still another object of this invention to provide a reactiveintercalating component having the formula

wherein each of n and m is independently an integer of 1 to 20, p is aninteger of 0 to 20, Ar₁ and Ar₂ are independently selected from thegroup consisting of ethidium bromide, acridine, mitoxantrone,oxazolopyridocarbazole, ellipticine and N-methyl-2,7-diazapyrenium, andderivatives thereof, and X is a reactive group.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows one embodiment of the polynucleotide delivery system of theinvention, where NLS is a nuclear localization sequence, MD is amembran-permeabilization component, and Ligand is a cell recognitioncomponent.

FIG. 2 shows the structure of gramicidin S.

FIG. 3 compares the efficiency of luciferase transfection withLipofectin™, pH-sensitive liposomes, and the gramicidin S/DOPE/DNAcomplex.

FIG. 4 shows the effect of gramicidin S to DNA ratio on transfectionefficiency.

FIG. 5 shows the effect of gramicidin S to DOPE ratio on transfectionefficiency.

FIG. 6 shows the effect of lipid type in the gramicidin S/lipid/DNAcomplex on transfection efficiency.

FIG. 7 shows the effect of substituting other peptides for gramicidin Sin the gramicidin S/lipid/DNA complex on transfection efficiency.

FIG. 8 shows a synthetic scheme for attaching targeting carbohydratesand/or reactive maleimide to spermidine bis-acridine.

FIG. 9 shows the basic scheme for coupling eptides to themaleimido-spermidine bis-acridine.

FIG. 10 shows a synthetic scheme for coupling to a degradable Lys-Lyspeptide bis-acridine.

FIG. 11 shows the results of the gel retardation assay of Example 3.

FIG. 12 shows the ability of several galactosyl bis-acridines to bringplasmid DNA into hepatocytes.

FIG. 13 shows a synthetic scheme for the trigalactosylated spermidinebis-acridine of Example 6.

DETAILED DESCRIPTION OF THE INVENTION

Definitions:

The term “polynucleotide” as used herein, includes RNA or DNA sequencesof more than one nucleotide in either single chain, duplex or multiplechain form. “Polynucleotide” is generic to polydeoxyribonucleotides(containing 2′-deoxy-D-ribose or modified forms thereof), i.e., DNA, topolyribonucleotides (containing D-ribose or modified forms thereof),i.e., RNA, and to any other type of polynucleotide which is anN-glycoside or C-glycoside of a purine or pyrimidine base, or modifiedpurine or pyrimidine base or abasic nucleotides. The polynucleotide mayencode promoter regions, operator regions, structural regions,termination regions, combinations thereof or any other geneticallyrelevant material.

The polynucleotides of the invention may also contain one or more“substitute” linkages as is generally understood in the art. Some ofthese substitute linkages are non-polar and contribute to the desiredability of the polynucleotide to diffuse across membranes. Otherscontribute to the increased or decreased biodegradability of thepolynucleotide. (Biodegradability will be affected, for example, byincreased or decreased nuclease sensitivity.) These “substitute”linkages are defined herein as conventional alternative linkages such asphosphorothioate or phosphoramidate, are synthesized as described in thegenerally available literature. Not all such linkages in the samepolynucleotide need be identical.

Modifications in the sugar moiety of the polynucleotide, for example,wherein one or more of the hydroxyl groups are replaced with halogen,aliphatic groups, or functionalized as ethers, amines, and the like, orwherein the ribose or deoxyribose is replaced with other functionallyequivalent structures, are also included. Modifications in the basemoiety include alkylated purines or pyrimidines, acylated purines orpyrimidines, or other heterocycles. Such “analogous purines” and“analogous pyrimidines” are those generally known in the art, many ofwhich are used as chemotherapeutic agents.

In particular, the sugar-phosphate backbone of the polynucleotide may bereplaced with a non-carbohydrate backbone such as a peptide or othertype of polymer backbone as discussed in P. E. Nielsen et al., Science(1991) 254:1497-1500.

The term “functional component” as used herein, includes DNA-maskingcomponents, cell recognition components, charge-neutralization andmembrane-permeabilization components, and subcellular-localizationcomponents.

The term “DNA-masking component”, as used herein, refers to a moleculecapable of masking all or part of the polynucleotide, thereby increasingits circulatory half-life by inhibiting attack by degrading reagents(such as nucleases) present in circulation.

The term “membrane-permeabilizing component”, as used herein, refers toany component that aids in the passage of a polynucleotide across amembrane. Thus, this term encompasses in part charge-neutralizingcomponents, usually polycations, that neutralize the large negativecharge on polynucleotides, and enable the polynucleotide to transversethe hydrophobic interior of a membrane. Many charge-neutralizingcomponents can act as membrane-permeabilizers. Membrane-permeabilizationmay also arise from amphipathic molecules.

A membrane permeabilizer is a molecule that can assist a normallyimpermeable molecule to traverse cellular membranes and gain entrance tothe cytoplasm of the cell. A membrane permeabilizer may be a peptide,bile salt, glycolipid, carbohydrate, phospholipid or detergent molecule.Membrane permeabilizers often have amphipathic properties such that oneportion is hydrophobic and another is hydrophilic, permitting them tointeract with membranes.

The term “liposome” as used herein, refers to small vesicles composed ofamphipathic lipids arranged in spherical bilayers. Liposomes are usuallyclassified as small unilamellar vesicles (SUV), large unilamellarvesicles (LUV), or multi-lamellar vesicles (MLV). SUVs and LUVs, bydefinition, have only one bilayer, whereas MLVs contain many concentricbilayers. Liposomes may be used to encapsulate various materials, bytrapping hydrophilic molecules in the aqueous interior or betweenbilayers, or by trapping hydrophobic molecules within the bilayer.

Liposomes exhibit a wide variety of characteristics, depending upontheir size, composition, and charge. For example, liposomes having asmall percentage of unsaturated lipids tend to be slightly morepermeable, while liposomes incorporating cholesterol or other sterolstend to be more rigid and less permeable. Liposomes may be positive,negative, or neutral in charge, depending on the hydrophilic group. Forexample, choline-based lipids impart an overall neutral charge,phosphate and sulfate based lipids contribute a negative charge,glycerol-based lipids are generally negatively-charged, and sterols aregenerally neutral in solution but have charged groups.

The term “cell recognition component” as used herein, refers to amolecule capable of recognizing a component on the surface of a targetedcell. Cell recognition components include: antibodies to cell surfaceantigens, ligands for cell surface receptors including those involved inreceptor-mediated endocytosis, peptide hormones, and the like.

The term “DNA-associating moiety” refers to a molecule or portionsthereof that interacts in a noncovalent fashion with nucleic acids.DNA-associating moieties include major- and minor-groove binders, whichare molecules thought to interact with DNA by associating with the majoror minor groove of double-stranded DNA. DNA associating moieties alsoinclude DNA intercalators, which are planar molecules or planar portionsof molecules thought to intercalate into DNA by inserting between andparallel to nucleotide base pairs. DNA associating moieties furtherinclude polycations, thought to associate with the negative charges onthe DNA backbone. When a single-stranded DNA or RNA is used as thetherapeutic strand, the complementary “linker strand” as describedherein may functionally act as the “DNA-associating moiety”.

DNA associating moieties may be covalently linked through a “reactivegroup” to a functional component of this invention. These reactivegroups are easily reacted with a nucleophile on the functionalcomponent. Such reactive groups (with their corresponding reactivenucleophiles) include, but are not limited to: N-hydroxysuccinimide(amine), maleimide and maleimidophenyl (sulfhydryl), pyridyl disulfide(sulfhydryl), hydrazide (carbohydrate), and phenylglyoxal (arginine).

The term “subcellular-localization component” as used herein, refers toa molecule capable of recognizing a subcellular component in a targetedcell. Recognized subcellular components include the nucleus, ribosomes,mitochondria, and chloroplasts. Particular subcellular-localizationcomponents include the “nuclear-localization components” that aid inbringing molecules into the nucleus and are known to include the nuclearlocalization peptides and amino acid sequences.

The Compositions:

The compositions of this invention in part are self-assemblingpolynucleotide delivery systems utilizing a polynucleotide incombination with one or more, preferably two or more of the followingfunctional components: DNA-masking components, cell recognitioncomponents, charge-neutralization and membrane-permeabilizationcomponents, and subcellular localization components. Each element inthis system is able to perform its indicated function and also becapable of assembling or disassembling with the polynucleotide asrequired. Individual elements of this system, and methods andintermediates for making these elements are also contemplated as part ofthis invention. One embodiment of the system is shown in FIG. 1.

The Polynucleotide

The polynucleotide may be single-stranded DNA or RNA, or double-strandedDNA or DNA-RNA hybrid. Triple- or quadruple-stranded polynucleotideswith therapeutic value are also contemplated to be within the scope ofthis invention. Examples of double-stranded DNA would include structuralgenes, genes including operator control and termination regions, andself-replicating systems such as plasmid DNA.

Single-stranded polynucleotides include antisense polynucleotides (DNAand RNA), ribozymes and triplex-forming oligonucleotides. This“therapeutic strand”, in order to have prolonged activity, preferablyhas as some or all of the nucleotide linkages stable, non-phosphodiesterlinkages. Such linkages include, for example, the phosphorothioate,phosphorodithioate, phosphoroselenate, or O-alkyl phosphotriesterlinkages wherein the alkyl group is methyl or ethyl.

For these single-stranded polynucleotides, it may be preferable toprepare the complementary strand to the therapeutic strand as part ofthe administered composition. This complementary strand is designatedthe “linker strand”, and is usually synthesized with a phosphodiesterlinkage so that it is degraded after entering the cell. The “linkerstrand” may be a separate strand, or it may be covalently attached to ora mere extension of the therapeutic strand so that the therapeuticstrand essentially doubles back and hybridizes to itself.

The linker strand may also have functionalities on the 3′ or 5′ end oron the carbohydrate or backbone of the linker that serve as functionalcomponents to enhance the activity of the therapeutic strand. Forexample, the phosphodiester linker strand may contain a targeting ligandsuch as a folate derivative that permits recognition and internalizationinto the target cells. If the linker is attached to its complementarytherapeutic strand that is composed of degradation-resistant linkages,the duplex would be internalized. Once inside the cell, the linker wouldbe degraded, releasing the therapeutic strand. In this manner thetherapeutic strand would have no additional functionalities attached andits function would not be impeded by non-essential moieties. Thisstrategy could be applied to any antisense, ribozyme or triplex-formingpolynucleotide. It would be used to deliver antiviral, antibacterial,antineoplastic, antiinflammatory, antiproliferative, anti-receptorblocking or anti-transport polynucleotides and the like.

A separate “linker strand” may be synthesized to have the directcomplementary sequence to the therapeutic strand and hybridize in aone-on-one fashion. Alternatively, the linker strand may be constructedso that the 5′ region of the linker strand hybridizes to the 5′ regionof the therapeutic strand, and the 3′ region of the linker strandhybridizes to the 3′ region of the therapeutic strand to form aconcatenate of the following structure

This concatenate has the advantage that the apparent molecular weight ofthe therapeutic nucleic acids is increased and its pharmacokineticproperties and targeting ligand:therapeutic oligonucleotide ratio can beadjusted to achieve the optimal therapeutic effect.

The Functional Components

DNA-Masking Components. The DNA-masking element of this system is amolecule capable of masking all or part of the polynucleotide, therebyincreasing its circulatory half-life by inhibiting attack by degradingreagents present in circulation.

In this invention, polyethylene glycol (PEG) can be covalently linkedwith a DNA-associating moiety by conventional methods as describedbelow, and used as a DNA-masking component. The PEG will have amolecular weight from about 700 to about 20,000 Daltons, preferablyabout 1800 to 6000 daltons, and is preferably present in a ratio(molecules PEG:bp DNA) from about 1:4 to 1:100, more preferably about1:20.

Alternatively, DNA may be masked through association with lipids. In oneembodiment, the DNA is encased in standard liposomes as described, forexample, in U.S. Patent No. 4,394,448 to Szoka et al., the specificationof which is hereby incorporated by reference. In another embodiment, theDNA is incubated with a synthetic cationic lipid similar to thosedescribed in U.S. Pat. No. 4,897,355 to Eppstein et al. These cationiclipids have the general formula

wherein n is an integer from 1 to 8, R¹ and R² are the same or differentand are alkyl or alkenyl having from 6 to 24 carbon atoms, R³ ishydrogen, alkyl or alkylamine having from 1 to 10 carbon atoms, and R⁴is a positively charged linear or branched alkyl or alkylamine havingfrom 1 to 30 carbon atoms, wherein one or more of the carbon atoms maybe substituted with NR′, wherein R′ is hydrogen, alkyl or alkylaminehaving from 1 to 10 carbons. Preferred groups that can function as the—N—R′ moiety are tris(aminoethyl)amine (NH₂CH₂CH₂)₃N, agmatine(decarboxyarginine) H₂N(CH₂)₄C(═NH)NH₂, 3-aminoethyl-1,3-propanediamineH₂N(CH₂)₃NH(CH₂)₂NH₂, 3-dimethylaminopropylamine (CH₃)₂NH(CH₂)₃NH₂,iminobis(N,N′)dimethylpropylamine NH((CH₂)₃N(CH₃)₂)₂,iminobis(3-aminopropyl)-1,3-propanediamine,1,4-bis(3-aminopropyl)piperazine, bis(propylamine) (NH₂(CH₂)₃)₂NH,spermidine, and spermine, wherein these groups are attached to the lipidmolecule through one of their nitrogen atoms.

In a specifically preferred embodiment, the synthetic cationic lipid isa synthetic cationic tail lipid having the formula

wherein n is an integer from 6 to 24, Y is selected from the groupconsisting of hydrogen, ethanolamine, choline, glycerol, serine andinositol, R¹ is alkyl or alkenyl having from 6 to 24 carbon atoms, R³ ishydrogen, alkyl or alkylamine having from 1 to 10 carbon atoms, and R⁴is a positively charged linear or branched alkyl or alkylamine havingfrom 1 to 30 carbon atoms, wherein one or more of the carbon atoms maybe substituted with NR′, wherein R′ is hydrogen, alkyl or alkylaminehaving from 1 to 10 carbons. Preferred groups that can function as the—N—R′ moiety are tris(aminoethyl)amine (NH₂CH₂CH₂)₃N, agmatine(decarboxyarginine) H₂N(CH₂)₄C(═NH)NH₂, 3-aminoethyl-1,3-propanediamineH₂N(CH₂)₃NH(CH₂)₂NH₂, 3-dimethylaminopropylamine (CH₃)₂NH(CH₂)₃NH₂,iminobis(N,N′)dimethylpropylamine NH((CH₂)₃N(CH₃)₂)₂,iminobis(3-aminopropyl)-1,3-propanediamine,1,4-bis(3-aminopropyl)piperazine, bis(propylamine) (NH₂(CH₂)₃)₂NH,spermidine, and spermine, wherein these groups are attached to the lipidmolecule through one of their nitrogen atoms.

It has been found that the above-described synthetic cationic lipidseffectively mask the DNA when associated therewith. Without attemptingto limit the invention in any way, it is believed that the lipids form amonolayer structure that encapsulates the DNA in some fashion.

Cell Recognition Components. The cell recognition element of this systemis a molecule capable of recognizing a component on the surface of atargeted cell, covalently linked with a DNA-associating moiety byconventional methods as described below. Cell recognition componentsinclude: antibodies to cell surface antigens, ligands for cell surfacereceptors including those involved in receptor-mediated endocytosis,peptide hormones, etc. Specific ligands contemplated by this inventioninclude: carbohydrate ligands such as galactose, mannose, mannosyl5-phosphate, fucose, sialic groups, N-acetylglucosamine or combinationsof these groups as complex carbohydrates such as those found onglycolipids of the blood groups or on various secreted proteins. Otherligands include folate, biotin, various peptides that can interact withcell surface or intracellular receptors such as the chemoattractantpeptide SEQ ID NO:2, peptides containing the arg-asp-glycine sequence orSEQ ID NO:3 peptides, peptides that contain a cystine residue or thatinteract with cell surface protein such as the human immunodeficiencyvirus GP-120, and peptides that interact with CD-4. Other ligandsinclude antibodies or antibody fragments such as described by A. Hertlerand A. Frankel, J Clin Oncol 7: 1932-1942. The specificity of theantibodies can be directed against a variety of epitopes that can beexpressed on cell surfaces including histocompatability macromolecules,autoimmune antigens, viral, parasitic or bacterial proteins. Otherprotein ligands include hormones such as growth hormone and insulin orprotein growth factors such as GM-CSF, G-CSF, erythropoietin, epidermalgrowth factor, basic and acidic fibroblast growth factor and the like.Other protein ligands would include various cytokines that work throughcell surface receptors such as interleukin 2, interleukin 1, tumornecrosis factor and suitable peptide fragments from such macromolecules.

Membrane-Permeabilizing Components. The membrane-permeabilizing elementof this system is a molecule that aids in the passage of apolynucleotide across a membrane. The liposomes and synthetic cationiclipids described above as DNA-masking components also may function asmembrane-permeabilization components.

The membrane-permeabilizing components of this invention also includepolycations that neutralize the large negative charge onpolynucleotides. Polycations of this invention include polylysine,polyarginine, poly (lysine-arginine) and similar polypeptides, and thepolyamines and the polycationic dendrimers. D. A. Tomalia et al., Agnew.Chem. Int. Ed. Engl. (1990) 29:138-175. Another class of polycations arethe cationic bile salts having the following formula:

wherein X and Y are independently H or OH, R³ is hydrogen, alkyl oralkylamine having from 1 to 10 carbon atoms, and R⁴ is a positivelycharged linear or branched alkyl or alkylamine having from 1 to 30carbon atoms, wherein one or more of the carbon atoms may be substitutedwith NR′, wherein R′ is hydrogen, alkyl or alkylamine having from 1 to10 carbons. Preferred groups that can function as the —N—R′ moiety aretris(aminoethyl)amine (NH₂CH₂CH₂)₃N, agmatine (decarboxyarginine)H₂N(CH₂)₄C(═NH)NH₂, 3-aminoethyl-1,3-propanediamineH₂N(CH₂)₃NH(CH₂)₂NH₂, 3-dimethylaminopropylamine (CH₃)₂NH(CH₂)₃NH₂,iminobis(N,N′)dimethylpropylamine NH((CH₂)₃N(CH₃)₂)₂,iminobis(3-aminopropyl)-1,3-propanediamine,1,4-bis(3-aminopropyl)piperazine, bis(propylamine) (NH₂(CH₂)₃)₂NH,spermidine, and spermine, wherein these groups are attached to the bilesalt through one of their nitrogen atoms.

In a different embodiment, the membrane-permeabilizing component of theinvention is an amphipathic cationic peptide. Amphipathic cationicpeptides are peptides whose native configuration is such that thepeptide is considered to have a cationic face and a neutral, hydrophobicface. In a preferred embodiment, the peptide is a cyclic peptide.Examples of the amphipathic cationic cyclic peptides of this inventionare gramicidin S (the structure of which is shown in FIG. 2), and thetyrocidines. The peptide may also contain some or all of the amino acidsin the D configuration as opposed to the naturally occurring Lconfiguration.

In a particularly preferred embodiment, the membrane-permeabilizingelement includes, in addition to the amphipathic cationic cyclicpeptides, either (1) a lipid, or (2) a simple polyamine, or both.

The lipid of the invention is an amphipathic molecule which is capableof liposome formation, and is substantially non-toxic when administeredat the necessary concentrations either in native form or as liposomes.Suitable lipids generally have a polar or hydrophilic end, and anon-polar or hydrophobic end. Suitable lipids include without limitationegg phosphatidylcholine (EPC), phosphatidylethanolamine,dipalmitoylphosphatidylcholine (DPPC), cholesterol (Chol),cholesterylphosphorylcholine, 3,6,9-tri-oxaoctan-1-ol-cholesteryl-3-ol,dimyristoylphosphatidylcholine (DMPC), and other hydroxy-cholesterol oraminocholesterol derivatives (see, e.g., K. R. Patel et al., BiochimBiophys Acta (1985) 814:256-64). The lipid is preferably added in theform of liposomes.

The added polyamine is preferably spermine or spermidine.

The membrane permeabilizing elements—the cyclic peptide and optionalphospholipid and polyamine—may be added to the compositionsimultaneously or consecutively. Preferably, the cyclic peptide is addedfirst, and the phospholipid or polyamine added later. The molar ratio ofadded cyclic peptide to added polyamine is preferably from about 1:1 toabout 1:3. The molar ratio of added cyclic peptide to added phospholipidis preferably from about 1:1 to about 1:20.

Subcellular-Localization Components. The subcellular-localizationelement of this system is a molecule capable of recognizing asubcellular component in a targeted cell, covalently linked with aDNA-associating moiety by conventional methods as described below.Particular subcellular components include the nucleus, ribosomes,mitochondria, and chloroplasts.

In a preferred embodiment of this invention, thesubcellular-localization component is a nuclear-localization component.The nuclear-localization components include known peptides of definedamino acid sequences, and longer sequences containing these peptides.One known peptide sequence is the SV 40 large T antigen heptapeptide SEQID NO:1. Other peptides include the influenza virus nucleoproteindecapeptide SEQ ID NO:4, and the adenovirus E1a protein sequence SEQ IDNO:5. Other sequences may be discerned from C. Dingwall et al., TIBS(1991) 16:478-481.

In another embodiment, the subcellular-localization component is alysosomal-localization component. A known component for targeting thelysosome is a peptide containing the sequence SEQ ID NO:6. In yetanother embodiment, the subcellular-localization component is amitochondrial-localization component. A known component for targetingmitochondria is a peptide containing the sequence SEQ ID NO: 7.

DNA-Associating Moieties

The DNA-associating moiety of this system refers to a portion of afunctional component that interacts in a noncovalent fashion withnucleic acids. The moiety is covalently linked to the rest of thefunctional component by conventional means or as described below.DNA-associating moieties are preferably major- and minor-groove binders,DNA intercalators, or general DNA binders. In the case ofsingle-stranded polynucleotides, the DNA-associating moiety may even bethe linker strand as described above. In such a case the functionalmoiety, such as the cell-recognition or subcellular-localizationcomponent is covalently linked to the linker strand.

In one preferred embodiment, the DNA-associating moiety is a major- orminor-groove binder. The major- and minor-groove binders are moietiesknown to associate or “lay in” the major or minor groove of DNA. Thesebinders include distamycin A and Hoechst dye 33258.

In another embodiment, the DNA-associating moiety is a nonspecific DNAbinder such as a polycation. Polycations of this invention includepolylysine, polyarginine, poly (lysine-arginine) and similarpolypeptides, and the polyamines and the polycationic dendrimers.

In another preferred embodiment, the DNA-associating moiety is a DNAintercalator. DNA intercalators are planar polycyclic molecules such asethidium bromide, acridine, mitoxantrone, oxazolopyridocarbazole,ellipticine and N-methyl-2,7-diazapyrenium, and derivatives thereof. Ina particular preferred embodiment, the intercalator is a dimerconsisting of two covalently linked planar polycyclic molecules. Aplanar polycyclic dimer moiety of this invention has the structure

wherein Z is a bond;

wherein each of n and m is independently an integer of 1 to 20, p is aninteger of 0 to 20,

Ar₁ and Ar₂ are independently selected from the group consisting ofethidium bromide, acridine, mitoxantrone, oxazolopyridocarbazole,ellipticine and N-methyl-2,7-diazapyrenium, and derivatives thereof.

The values of n and m are important as they determine the spacing of theintercalated acridine monomers in the DNA. More preferred values of nand m are 3 and 4, respectively. Bis-acridine dimers, wherein Ar₁ andAr₂ are both acridine, are preferred.

This preferred DNA-associating moiety will be covalently attached to afunctional moiety, said moiety being a cell recognition moiety,subcellular localization moiety, or membrane permeabilizing moiety asdescribed above. The value of p determines the separation of theintercalator from the functional moiety. Preferred values for p are from0 to 8.

The DNA-associating moiety may be covalently attached to multiple copiesof, or more than one functional moiety. For example, the bis-acridinedimer may be attached to three galactose residues that bind to thehepatocyte asialoorosomucoid receptor (See Compound 26 in FIG. 13).

A preferred method for attaching the DNA-associating dimer to thefunctional moiety involves a precursor having the formula

wherein each of n and m is independently an integer of 1 to 20, p is aninteger of 0 to 20,

Ar₁ and Ar₂ are independently selected from the group consisting ofethidium bromide, acridine, mitoxantrone, oxazolopyridocarbazole,ellipticine and N-methyl-2,7-diazapyrenium, and derivatives thereof; and

X is a reactive group selected from the group consisting ofN-hydroxysuccinimide, maleimide, maleimidophenyl, pyridyl disulfide.,hydrazide, and phenylglyoxal.

In a preferred embodiment, Ar₁ and Ar₂ are acridine, p is 3 and X isp-maleimidophenyl. This intercalating moiety is then coupled to thefunctional moiety through a sulfhydryl group on the functional moiety,for example, to obtain a bifunctional component having the structure

wherein

Y is a functional component;

each of n and m is independently an integer of 1 to 20, p is an integerof 0 to 20,

Ar₁ and Ar₂ are independently selected from the group consisting ofethidium bromide, acridine, mitoxantrone, oxazolopyridocarbazole,ellipticine and N-methyl-2,7-diazapyrenium, and derivatives thereof; and

X is a reactive group selected from the group consisting ofN-hydroxysuccinimide, maleimide, maleimidophenyl, pyridyl disulfide,hydrazide, and phenylglyoxal.

Biodegradable linkers such as peptides having the sequence -lys-lys- mayalso be used in attaching the functional component to the intercalator.

In yet another embodiment of this invention, the planar polycyclic dimerhas the formula

wherein

Ar₁ and Ar₂ are independently selected from the group consisting ofethidium bromide, acridine, mitoxantrone, oxazolopyridocarbazole,ellipticine and N-methyl-2,7-diazapyrenium, and derivatives thereof;

each aa is independently an amino acid;

x and z are integers independently selected from 1 to 100;

y is an integer from 0 to 5;

aa₁ and aa₂ are lysine residues;

N¹ and N² are nitrogens from the ε-amino groups of lysine residues aa₁and aa₂.

Utility of the Polynucleotide Delivery System

The polynucleotide delivery system of the invention is useful in atherapeutic context. In therapeutic applications, the system of theinvention can be formulated for a variety of modes of administration,including systemic and topical or localized administration. Techniquesand formulations generally may be found in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., latest edition.

For systemic administration, parenteral administration such as injectionis preferred, including intramuscular, intravenous, intraperitoneal, andsubcutaneous. For treating disorders of the lung, administration of thepolynucleotide delivery system is done by inhalation or installation ofthe system directly into the lung.

For injection, the systems of the invention are formulated in liquidsolutions, preferably in physiologically compatible buffers such asHank's solution or Ringer's solution. In addition, the systems may beformulated in solid form and redissolved or suspended immediately priorto use. Lyophilized forms are also included.

Systemic administration can also be by transmucosal or transdermalmeans, or the systems can be administered orally, or through intranasalor inhaled aerosols. For transmucosal or transdermal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art, andinclude, for example, for transmucosal administration bile salts andfusidic acid derivatives. In addition, detergents may be used tofacilitate permeation. Transmucosal administration may be through nasalsprays, for example, or using suppositories. For oral administration,the systems are formulated into conventional oral administration formssuch as capsules, tablets, and tonics.

For topical administration, the systems of the invention are formulatedinto ointments, salves, gels, or creams, as is generally known in theart.

The following examples are meant to illustrate, but not to limit theinvention.

EXAMPLE 1 Gramicidin S Transfection

Lipofectin™ is a synthetic cationic lipid,N-[1(-2,3-dioxeoyloxy)propyl]-N,N,N-trimethylammonium chloride, (DOTMA)in combination with phosphatidylethanolamine to form a charge complexwith the negatively charged DNA. This complex is thought to fuse withthe cell membrane and deliver DNA into the cytoplasm. An alternativeapproach uses pH sensitive liposomes composed of a negatively chargedlipid and phosphatidylethanolamine. C. Y. Wang et al., Biochem (1989)28:9508-9514. The delivery mechanism involves endocytosis of theliposome, as the pH in the endosome becomes acidic, the liposomalbilayer destabilizes and fuses with the endosomal membrane. The contentsof the liposome are then introduced into the cytoplasm of the cell.C.-J. Chu et al., Pharmaceut Res (1990) 7:824-834.

We have compared Lipofectin™ to a pH-sensitive cholesterylhemisuccinate(Chems)/phosphatidylethanolamine (PE) liposome composition and togramicidin S/dioleoylphosphatidylethanolamine (DOPE)/DNA complexes forthe delivery and expression of DNA in mammalian cells. Plasmidscontaining strong promoters and either firefly luciferase or βgalactosidase were used as indicators for gene transfer.

Cell Transfection Protocol.

CV-1, p388D1, HepG2 and HeLa cells were provided by the UCSF CellCulture Facility. The Lipofectin™ reagent was used as described in theproduct insert (Gibco-BRL, Gaithersburg, Md.). KD83 cells were obtainedfrom DNAX (Palo Alto, Calif.). Cells were plated at a density of0.5-1×10⁶ cells per 60 mm dish and grown 16 to 20 hrs at 37° C. under 5%CO₂ in appropriate media containing 10% fetal calf serum (FCS). Prior toincubation either with liposomes, Lipofectin™, or the gramicidinS/DOPE/DNA complex, cells were washed once with 2 ml of FCS-free DMEH-21 medium. The transfection system was then added in 2 ml of the samemedia. In some experiments, transfection took place in 10% FCScontaining DME H-21. After 5 hrs. media was removed and replaced by 3 mlof appropriate media with 10% FCS. Luciferase activity was measuredafter 48 hrs as described (A. R. Brasier et al., Biotechniques (1989)7:1116-1122). Briefly, cells were washed twice with ice-cold phosphatebuffer saline without Ca²⁺ and Mg²⁺ (PBS), treated with 400 μl of 25 mMglycylglycine (pH 7.8) in lysis buffer (containing 1% Triton) andscraped. After centrifugation, 100 μl of supernatant were mixed with anoptimal amount of 50 mM ATP. D-luciferin (Sigma, 100 μl of a 1 mMsolution) was then injected and the emitted light was integrated duringthe first 10 sec. using a bioluminometer (Bioluminescence AnalyticalLaboratories Inc., San Diego, Calif.). Proteins in the supernatant wereassayed using the technique of Bradford (Bio-Rad kit). Results wereexpressed as light units per mg of cell-protein.

Luciferase Assay

In order to compare the potency of three different viral luciferase genepromoters, RSV, SV40 and CMV, we have transfected several mammalian celllines with the corresponding Lipofectin™ complexed-plasmids. Each dishof cells received 2 μl of plasmid combined with 10 μl of Lipofectin™ asdescribed above. Promoter strength was estimated by the luciferaseexpression at 48 hr given by the corresponding plasmid. The CMV promoter(pCLuc4 plasmid) led to the highest luciferase expression in HeLa, HepG2and p388D1 cells, while SV40 promoter (pSV2 plasmid) was more potent inCV-1 cells. Therefore for further experiments, pSV2 plasmid has beenused in CV-1 cells and pCLuc4 in other cell-lines.

Liposome characterization

Plasmid encapsulation efficiency was determined after separation ofencapsulated from non-encapsulated plasmid on Ficoll gradients. About22±3% of the total DNA added was encapsulated. Liposome diameters,measured by dynamic light scattering and were 372±38 nm, 295±65 nm and464±20 nm for DOPE/CHEMS, DOPC/CHEMS and PS/Chol liposomes respectively(results are the mean±SD of three independent light scatteringdeterminations).

A. Gramicidin S and Phosphatidylethanolamine

Typical complex preparation was made by diluting 20 μg of plasmid DNA in300 μl of 30 mM Tris Cl pH 9 in a polystyrene tube. Gramicidin S wasdiluted in 30 mM pH 9 Tris Cl buffer to a concentration of 2 mg/ml froma stock solution at 20 mg/ml in DMSO. 20 μl of diluted gramicidin S(i.e. 40 μg) solution was added to the DNA and quickly mixed. Then 170nmoles of liposomes were added slowly drop by drop to the DNA/gramicidinS mixture. Liposomes were prepared by drying 4 μmoles of lipids undernitrogen with a rotavapor and by rehydrating the film with 4 ml of 30 mMpH 9 Tris Cl buffer. Liposomes were subsequently sonicated 30 min underargon using a bath sonicator. The diameter of the complex was determinedby dynamic light scattering. Other peptides including tyrocidine (U.S.Biochemicals), polymyxin B (Sigma) and polylysine 100 (Sigma) and thepolycationic Starburst™ dendrimer (Polyscience, Inc.) were also used toform the complex with DNA and lipids.

The efficiency of transfection was monitored by measuring the expressionof luciferase in Cv-1 cells as described above. The dose responsecomparing the amount of DNA added in the three transfection systems isillustrated in FIG. 3. Light units per mg cell protein in a log scaleare plotted on the Y axis and the amount of DNA added on the X axis. Theopen boxes designate results using the Gramicidin S-dioleoylphosphatidylethanolamine-DNA complex. This complex induces a 10-fold greater levelof expression than obtained with Lipofectin, and a 1000- to 10,000-foldgreater level of expression than obtained using the pH sensitiveliposomes.

B. Gramicidin S-DNA Ratio Effects

The gramicidin S-DOPE-DNA complex was prepared as described in Example1-A except the amount of gramicidin S added to the complex was varied atconstant amounts of DNA (20 ug) and DOPE (170 nmoles). The complex wasadded to CV-1 cells and the luciferase activity measured as described inExample 1. The result is presented in FIG. 4 and illustrates thatmaximum expression using the gramicidin S-DOPE-DNA complex occurs whenthe charge on the DNA is neutralized by the charge on the gramicidin.

C. Lipid Concentration Effects

The gramicidin S-DOPE-DNA complex was prepared as described in Example 1except the amount of DOPE added to the complex was varied at constantamounts of DNA (20 ug) and gramicidin S (40 μg). The complex was addedto CV-1 cells and the luciferase activity measured as described inExample 1. The result is presented in FIG. 5, which illustrates that inthe absence of the DOPE, expression is low. Maximum expression using thegramicidin S-DOPE-DNA complex occurs when the ratio of DOPE togramicidin S is above 5/1:mole/mole.

D. Lipid Type Effects

The gramicidin S-lipid-DNA complex was prepared as described in Example1 except the type of phospholipid added to the complex was varied atconstant amounts of DNA (20 ug) and gramicidin S(40 ug). The lipidcompositions employed were DOPE; DOPE: dioleoylphosphatidylcholine(DOPC):2/1, palmitoyloleoylphosphatidylethanolamine (POPE), monomethylDOPE (mmDOPE); dimethyl DOPE (dm DOPE); DOPC anddipalmitoylphosphatidylethanolamine (DPPE). The complex was added toCV-1 cells and the luciferase activity measured as described inExample 1. The result is presented in FIG. 6, which illustrates thatexpression of luciferase activity is maximal with DOPE or a mixture ofDOPE/DOPC:2/1 in the complex. Luciferase activity is appreciablydiminished when the amino group on the DOPE is substituted with 2 (dmDOPE) or 3 methyl groups (DOPC). Expression of the encoded gene is alsoappreciably reduced when DPPE is used. This latter lipid has saturatedacyl chains and a high transition temperature, which means the acylchains of DPPE are less fluid than the other lipids tested in thisseries.

E. Effects of Added Non-Amphipathic Positively Charged Spermidine

The data presented in Example 2 show that gene expression due to thegramicidin S-DOPE-DNA complex is maximal when the negative charges onDNA are neutralized by the positive charges on gramicidin. To determinewhether charge neutralization or membrane permeabilization is moreimportant for gene transfer using this system, the positive chargecontribution from gramicidin S was incrementally replaced by thepositively charged polyamine, spermidine. The gramicidin S-lipid-DNAcomplex was prepared as described in Example 1 except the amount ofgramicidin S added to the complex was varied at constant amounts of DNA(20 ug). The requisite positive charges required to neutralize the DNAwas supplied by spermidine. The complex was prepared with or without 170nmoles of DOPE. The complex was added to CV-1 cells and the luciferaseactivity measured as described in Example 1. The results are given inTable 1 below, with luciferase activity expressed as light units/mg cellprotein. The first activity was always greater when DOPE was present inthe complex. In the absence of DOPE, the sequential replacement ofpositive charge due to gramicidin S by spermidine leads to a biphasicresponse. The expression of luciferase initially increased to a valueabout 100 fold less than the maximal response obtained in the presenceof DOPE. When the percent of charge neutralization due to gramicidin Sdropped below 25% transfection activity was totally lost. Thus, membranepermeabilization function of gramicidin S is more important than thecharge neutralization function.

TABLE 1 Spermidine Charge Neutralization % charges brought by GS w/olipids with lipids 100 4.5 ± 2 10³ 8.5 ± 0.7 10⁸ 75 4 ± 2.5 10⁵ 5 ± 210⁸ 25 2 ± 2.5 10⁶ 2 ± 0.5 10⁷ 12.5 0 2 ± 0.5 10⁷

F. Use of Other Positively Charged Peptides

The peptide-DOPE-DNA complex was prepared as described in Example 1except the type of peptide added to the complex was varied at constantamounts of DNA (20 ug) and DOPE (170 nmoles). The peptides employed werepolymyxin B, a cyclic cationic peptide; polylysine, a linear cationicpeptide; tyrocidine, a cyclic cationic peptide with a similar structureto gramicidin S but containing only a single positive charge andgramicidin S. The luciferase plasmid was also transfected into the cellsusing Lipofectin™. The complex was added to CV-1 cells and theluciferase activity measured as described in Example 1. FIG. 7 showsthat gramicidin S induced the greatest level of expression followedclosely by the related cyclic peptide tyrocidine. Both cyclic peptideswere superior to Lipofectin™ at transferring the DNA into cells.Activity was also seen with the other two peptides, polymyxin B andpolylysine, however, the level of luciferase expression mediated bythese two cationic peptides was inferior to that induced by gramicidin Sor tyrocidine.

G. Comparison of DNA-Dendrimer Complex and DNA-PolylysineComplex-Mediated Transfections

To find better chemically-defined alternatives to the polyamine polymerssuch as polylysine, we have employed the hydrophilic branched polycationmacromolecules also know as the Starburst™ Dendrimer microparticles,Tomalia et al., supra, to form a complex with DNA or with DNA and thepermeabilizing amphipathic peptide GALA SEQ ID NO:10. R. Parente et al.,Biochemistry (1990) 29:8720-8728. The complex was prepared by diluting12 μg of pCLuc4 plasmid in 660 μl of HBS (20 mM Hepes, 150 mM NaCl, pH7.4) in a polystyrene tube. Polylysine (Sigma Chemical Co.) orStarburst™ Dendrimer microparticles of the fifth generation (1 nmole)(Polysciences, Inc.) was dissolved in 340 μl of HBS and added slowly(dropwise) to the DNA solution. In these conditions, the positivecharges from the epsilon amino groups of the polylysines or from theperipheral amines of the dendrimers are in 1.3-fold excess over thenegative charges of the plasmids. When the peptide GALA SEQ ID NO:10 wasadded, it was added so that the negative charges on GALA SEQ ID NO:10neutralized the excess charges on the dendrimer. The mixture was left tostand for thirty minutes after the last addition at room temperature andthen 500 μl of the mixture was added to CV-1 cells. The transfectionprotocol was carried out as described above. In this experiment, thebest transfection protocol was accomplished with the GALA SEQ IDNO:10-dendrimer-DNA complex, followed by the dendrimer-DNA and then bypolylysine-DNA. The results are shown in Table 2 below.

TABLE 2 DNA-Dendrimer Mediated Transfection Luciferase lights Condition(units per mg cell protein) Dendrimer-GALA SEQ ID NO:11-DNA (9 ± 2) ×10⁵ (n = 2) Dendrimer-DNA (5 ± 2) × 10⁵ (n = 2) Polylysine-DNA (2.7 ±0.1) × 10⁵ (n = 2)

EXAMPLE 2 Synthesis of Reactive and Functionalized SpermidineBis-Acridines

Spermidine bis-acridine derivatives (synthesis shown in FIG. 8)intercalate into double stranded nucleic acids with affinity constantsgreater than 1×10⁴ (pH 7.4; 0.2M NaCl) and can be used to attach avariety of targeting molecules to DNA. Carbohydrates, peptides,hormones, vitamins, cofactors, proteins or antibodies can all be used astargeting ligands.

A. Spermidine Bis-Acridine

The scheme for directing nucleic acids to certain sites of the body isbased upon the intercalation of a ligand which interacts with a cellsurface component into the double stranded DNA. A procedure forselective N⁴-acylation of spermidine, using N¹,N⁸-bis(t-butoxycarbonyl)spermidine as starting material, has been reported. R. J. Bergeron etal., Synthesis (1982) 689-692. We have used this procedure (FIG. 8) tolink the acid functionalized galactosyl derivatives 9(n=1) and 9′(n=4)to the secondary amino group of N¹,N⁸-tBoc-protected spermidine (15) andthe resulting galactosylated spermidines 17 and 17′ were, afterdeprotection, further alkylated with 9-phenoxyacridine by a standardchemical procedure to transform them into bis-intercalator compounds 21and 21′. The synthesis of the carboxylic acid functionalized galactosylderivatives is detailed (J. Haensler et al., Biochim Biophys Acta (1988)946:95-105) and is easily applicable to a wide range of carbohydrateligands (M. M. Ponpipom et al., J Med Chem (1981) 24:1388-1395). Thetitle compounds were obtained in an overall yield of 30% and the NMR andmass spectrometry data are consistent with the proposed structure.

B. Activated Spermidine Bis-Acridine

Based upon the above scheme, a versatile method for attaching peptidesto a spermidine bis-acridine derivative, has been developed.N¹,N⁸-bis(t-butoxycarbonyl) spermidine (15) was N⁴-acylated withN-succinimidyl-4-(p-maleimidophenyl)butyrate (SMPB) (4), deprotected andcoupled to acridine to make a bis-intercalator bearing a maleimide group(20). A single compound was obtained after chromatographic purificationon silica gel in 25% overall yield. The NMR and mass spectrometryresults are consistent with the assigned structure.

C. Spermidine Bis-Acridine Linked to a NLS

The NLS peptide SEQ ID NO:1 (Kaneda et al., supra, Science (1989)243:375-378) and control peptides with the same composition but adifferent sequence have been synthesized on an ABI automatic peptidesynthesizer with an N-terminal cysteine residue. The cysteine peptide isthen attached to the maleimide bearing intercalator (FIG. 9) and can beanchored into double stranded nucleic acids.

D. Biodegradable Linkers

Biodegradable linkers consisting of a lys-lys peptide linkage aresynthesized in the manner. shown in FIG. 10. In the figure, a galactoseresidue is placed on the unprotected amine. Alternatively, a protectedpeptide containing two adjacent lysine residues is synthesized by solidphase synthesis. The peptide carries membrane permeabilization functionsor targeting functions and acridine residues are added to the twoε-amino groups on the lysines.

EXAMPLE 3 Gel Retardation Assay of pCLuc4 Plasmid with GalactosylatedIntercalators and Agglutinin

To demonstrate that the galactosylated bis-acridines 21 and 21′ ofExample 2 (21′ is the homolog of 21 where the galactose is separatedfrom spermidine bis-acridine by three extra carbons) can interact with asoluble receptor while attached to DNA, we used a gel shift assay. Inthis assay, a galactose binding protein, Ricinus Communis lectin RCA₁₂₀,was incubated with the galactosyl-bis-acridine-DNA complex. If thisprotein interacts with the complex and the complex remains associatedwith the DNA, the DNA does not migrate into the electrophoresis gel.Each sample of the plasmid pCLuc4 (2 μl; 140 ng) was mixed with 13.5pmoles of 21 or 21′ and then 1 μl (33.3 pmoles) of RCA₁₂₀ was added,plus when indicated, an excess of free galactose (1.35 nmoles). After 30minutes of incubation at room temperature, the samples wereelectrophoresed through a 0.8% agarose gel using a 0.04 M Tris-Acetatebuffer system (pH 7.6) and stained with ethidium bromide to visualizethe DNA (FIG. 11).

Intercalation of the galactosylated spermidine bis-acridines into thepCLuc4 plasmid is shown by the retardation observed for the plasmid whencomplexed with compounds 21′ (lane B) or 21 (lane E). Intercalation ofthe bis-acridine into the DNA produces a change from the supercoiledform to a relaxed circular form, which migrates slower.

The capability of the plasmid-galactose complex to bind to a solublereceptor for galactose is shown by the almost complete retardation ofthe complex in presence of Ricinus Communis lectin RCA₁₂₀ (lane C andF). RCA₁₂₀ is a dimer and has two binding sites selective for terminalβ-D-galactosyl residues and thus too can crosslink the plasmid-galactosecomplexes. The interaction of RCA₁₂₀ with the plasmid pCLuc4 whencomplexed to compounds 21 or 21′ results in a formation of largeaggregates which do not penetrate into the gel. This interaction appearsto be much more efficient when the plasmid is complexed with 21′ thanwith 21. To crosslink the plasmids, RCA₁₂₀ has to overcome electrostaticrepulsions existing between adjacent plasmids. Thus, separating thegalactose from the surface of the plasmids by a spacer arm, as in caseof the complexes obtained with compound 21′, makes the binding of thelectin easier. As a result of a multivalent interaction, the plasmidaggregates formed by RCA₁₂₀ are very stable and are not dissociated by a100-fold excess of a competing monovalent ligand such as galactose(lanes D and G).

EXAMPLE 4 Binding of Bis-acridines to Double-Stranded DNA Using EthidiumBromide Displacement Assay

The affinity of the bis-acridines for calf thymus DNA was calculatedfrom the displacement of ethidium bromide from double stranded nucleicacids (Nielsen, supra). Ethidium displacement was monitored by thedecrease of the ethidium bromide fluorescence (ex.=540 nm, em.=610 nm)that occurs when it is released from DNA. The association constants ofthe bis-acridines relative to ethidium bromide are calculated from theirIC₅₀. In this study, spermidine bis-acridine trihydrochloride (SBA•3HCl)synthesized as described (Nielsen, supra), was used as the referencecompound. As a result of the loss of one of its three positive charges,a slight but significant decrease in affinity is observed when the N⁴amino group of spermidine bis-acridine is engaged in an amide bond withthe targeting carbohydrate in compound 21 (Gal-bA•2 HCl). However, wepredict an increase in affinity when spermidine bis-acridine is linkedto the highly positively charged NLS peptide SEQ ID NO:1. G. Karup etal., Int J Peptide Protein Res (1988) 32:331-343.

The binding constants of the various bis-acridine conjugates synthesizedto attach targeting ligands to DNA in the various examples are given inTable 3.

TABLE 3 Dissociation Constants of the Bis-acridines from Calf Thymus DNA(in M) SBA · 3HCl 2.4 × 10⁻⁸ Gal-3-bA¹ 3.5 × 10⁻⁷ Gal-6-bA² 7.9 × 10⁻⁷Gal₃Lys₂-bA³ 5.4 × 10⁻⁶ Maleimido-bA⁴ 6.5 × 10⁻⁷ WTcys-bA⁵ 1.4 × 10⁻⁷SNL-bA⁶ 1.4 × 10⁻⁷ ¹Compound 21 where n = 3 ²Compound 21 where n = 6³Compound 26 (shown in Example 6) ⁴Compound 20 ⁵SBA linked to SEQ IDNO:8 ⁶SBA linked to SEQ ID NO:9

The binding constants for the various bis-acridines are computed from anethidium bromide displacement assay by using a method to determine thebinding affinity of a 4-Mer for a linear lattice via noncooperativecompetitive binding with a 2-Mer (A. Wolfe and T. Meehan. J.Mol.Biol.223, 1063-1087, 1992) and an intrinsic dissociation constant of5.3×10⁻⁶M for ethidium bromide.

EXAMPLE 5 Ability of Bis-acridine Galactosyl Ligands to Target DNA toCell-surface Receptors

To demonstrate the factors that control targeting ability of thebis-acridine intercalators containing a galactosyl targeting ligand, rathepatocytes were isolated from rat liver and placed in culture at adensity of 10⁶ hepatocytes in 60 mm petri dishes in 3 ml of minimumessential medium (MEM) medium containing 5% fetal calf serum andantibiotics. The hepatocytes are shown to have galactose receptors bybinding asialoorosomucoid. After 18 hours at 37° C., the medium isremoved and replaced with 1 ml of MEM. Then 1 ug of ¹²⁵I-labeled plasmidDNA complexed to either SBA•3HCL, Gal-3-bA, Gal-6-bA or Gal₃-Lys₂-bA in100 ul water was added to the culture dish. The intercalator to plasmidratio was 500:1 or 1000:1. The cells were incubated for an additionalhour at 37° C., then rinsed and the protein digested in 1 ml NaOH (1N).The cell lysate was counted for radioactivity and the protein measured.The amount of cell associated plasmid is expressed as ng of plasmid permg of cell protein and graphed as a function of complexing agent (FIG.12). Although all three galactosyl bis acridine compounds bind to DNA(Table 3) and can interact with a soluble galactose binding protein(Example 3), only the Gal₃-Lys₂-bA was able to interact with the cellsurface receptor. Thus, efficient targeting to cell surface receptorsrequires a longer spacer arm and/or a cluster of galactosyl residues asprovided by the Gal₃-Lys₂-bA (synthesis shown in FIG. 13 and Example 6).

EXAMPLE 6 Synthesis of a Biodegradable Bis-acridine Containing ThreeTargeting Ligands: Trigalactosylated Spermidine Bis-acridine

The complete synthesis of this molecule is shown in FIG. 13.

Synthesis of L-Lysyl-L-Lysine bis-trifluoroacetate(22): Nε-tBOC-L-Lysine(603 mg, 2.45 mmol) and Nα,Nε-bis-tBOC-L-Lysine-p-nitrophenyl ester(2.28 g; 4.9 mmol) were mixed in 40 ml of N-methyl morpholine containing640 μl of N,N diisopropylethylamine (3.7 mmol). The mixture was stirredovernight at room temperature under argon filtered to remove insolubletraces of unreacted Nε-tBOC-L-Lysine and evaporated to dryness underhigh vacuum. The residue was purified in a silica gel column eluted withthe system CHCl₃/CH₃OH/H₂O 9:1:0.1, to afford 1.22 g of puretBOC-protected Lysine dimer; yield 87%

Deprotection: To a cooled flask (dry ice) containing 700 mg (1.2 mmol)of the tBOC-protected Lysine dimer were added 5 ml of TFA. The mixturewas warmed to room temperature and stirred under argon. After 30 minutesstirring the trifluoroacetic acid was evaporated in vacuo. The residuewas taken up in acetone and evaporated (5 times). Finally the residuewas redissolved in 14 ml of water, washed three times with 8 ml ofchloroform and the residue was lyophilized to give 480 mg of the titlecompound; yield 80%.

Protected trigalactosyl lysine dimer: Synthesis, of Nα-[Nα,Nε-Bis[6-(1-thio-2,3,4,6,-tetra-O-acetyl-β-D-galactopyranosyl)hexanoyl]-L-Lysyl-Nε-[6-(1-thio-2,3,4,6,-tetra-O-acetyl-β-D-galactopyranosyl)hexanoyl]-L-Lysine(23).

To a solution of L-Lysyl-L-Lysine bis-trifluoroacetate (400 mg; 0.8mmol) in 8 ml of anhydrous DMF containing 505 μl of triethylamine (3.6mmol) was added p-nitrophenyl6-(1-thio-2,3,4,6,-tetra-O-acetyl-β-D-galactopyranosyl)hexanoate (1.44g; 2.4 mmol). The mixture was stirred overnight under argon andevaporated to dryness. The residue was purified by chromatography on asilica gel column eluted with CHCl₃/CH₃OH/H₂O 90:10:0.5, to give 463 mgof the title compound; yield 35%.

MS: Calculated for C₇₂H₁₀₉N₄O₃₂S₃ m/z=1653, found m/z=1653.6 (M+H)+,m/z=1677.6 (M+Na)+, m/z=1693.6 (M+K)+.

Reaction with selectively blocked spermidine:N4-(Nα-[Nα,Nε-Bis[6-(1-thio-2,3,4,6,-tetra-O-acetyl-β-D-galactopyranosyl)hexanoyl]]-L-Lysyl-Nε-[6-(1-thio-2,3,4,6,-tetra-O-acetyl-β-D-galactopyranosyl)hexanoyl]-L-Lysyl]-N1,N8-bis-tBOC-spermidine.(24).

Compound 23 (132 mg; 80 μmol) was activated by esterification withN-Hydroxysuccinimide (11 mg, 96 μmol) in the presence ofN,N′-dicyclohexylcarbodiimidide (DCC) (20 mg; 97 μmol) in 5 ml ofanhydrous methylene chloride. After 4 h stirring at room temperatureunder argon, the urea precipitate was removed by filtration and thefiltrate was evaporated in vacuo. The dry residue was redissolved in 3ml of acetonitrile and added dropwise to a solution ofN1,N8-bis(t-butoxycarbonyl spermidine) Hydrochloride (30 mg; 80 μmol) in3 ml of acetonitrile containing 14 μl of triethylamine (100 μmoles). Themixture was further stirred for 48 h at room temperature under argon,evaporated in vacuo to a residue which was purified on a silica gelcolumn, eluted with CHCl₃/CH₃OH/H₂O 90:10:1, to afford 71 mg of thetitle compound; yield 45%.

Deprotection: Synthesis ofN4-[Nα-[Nα,Nε-bis[6-(1-thio-β-D-galactopyranosyl)hexanoyl]]-L-Lysyl-Nε-[6-(1-thio-β-D-galactopyranosyl)hexanoyl]]-L-Lysyl]spermidine.(25) Compound 24 (71 mg; 36 μmol) was deprotected asdescribed previously for compound 17 (See Example 2.A). The tBOCprotecting groups were removed from the spermidine linker by treatingwith 5 ml of TFA for 30 min and the acetyl protecting groups wereremoved from the galactosyl headgroups by treating overnight with amixture of CH₃OH/NEt₃/H₂O 5:4:1. The bis-trifluoroacetate salt of thespermidine derivative was converted to the free amine by passing a watersolution of the salt through a small BIO-RAD AG 1×2 (OH⁻) column. Thefractions positive for carbohydrates and for amines were pooled togetherand lyophilized to give 36 mg of compound 25); yield 79%.

Acridine attachment: Synthesis ofN4-[Nα-[Nα,Nε-bis[6-(1-thio-β-D-galactopyranosyl)hexanoyl]]-L-Lysyl-Nε-[6-(1-thio-β-D-galactopyranosyl)hexanoyl]]-L-Lysyl)-N1,N8-bis-acridinespermidine.(26) (“Gal₃-Lys₂-bA”) Compound 25 (36 mg; 28.5 μmol) and 18mg of 9-phenoxyacridine were dissolved in 3 g of phenol at 80° C. andthe solution was further stirred for 2 h at 80° C. under argon. Themixture was then cooled to about 40° C. and poured into 15 ml of etherto precipitate the aminoacridines. The yellow precipitate was collectedby filtration on a filter paper and redissolved in 4 ml of abutanol/methanol mixture 3:1. This solution was then concentrated byevaporation to about 1 ml and the bis-acridine derivative was isolatedby chromatography on a silica gel column, eluted withn-Butanol/Pyridine/Acetic acid/Water 6:2:1:2.

34 mg of the title compound are obtained; yield 21%. MS: Calculated forC₈₁H₁₁₇N₉O₂₀S₃ m/z=1631, found m/z=1632.8 (M+H)+, m/z=1654.8 (M+Na)+.

EXAMPLE 7 Transfection Assay Using Nuclear Localization Sequences

5 μl of Tris-EDTA(TE) containing a trace amount of a 5 Kb radioiodinatedplasmid (CMV-βGal) and 50 μl of water containing 8 nmoles of the nuclearlocalization peptide-bis-acridine conjugate of Example 2-C were added to80 μg of pCLuc4 (123 neq. bp) in solution in 45 μl of TE buffer (pH 8).The ratio of plasmid to peptide conjugate was 1:300. After 1 hourstanding at room temperature 100 μl of Tris-Cl buffer (pH 9) was addedto the complex and the resulting solution was mixed with 12 pmoles oflipids (DOPE/CHEMS 2:1, molar ratio) dissolved in 600 μl of ether forthe preparation of pH-sensitive liposomes.

The vesicles containing the DNA-peptide complexes were separated fromnonencapsulated material by floating the liposomes through a Ficollgradient. Encapsulation efficiency (20%±4%) was determined by dynamiclight scattering (Coulter N4, Coultronics).

Cells were transfected with 4 μg of liposome-encapsulated plasmid (100μl of the liposome solution) for 5 hours at 37° C. and luciferaseactivity was counted after 48 hours in a bioluminometer. Table 4 showsthe measured light units/mg cell protein as a function of the liposomalcontent. The values are the averages of three determinations.

TABLE 4 Liposomal Content Plasmid-WTcys-bA Plasmid-SNL-bA Plasmid AloneComplex Complex (0.32 ± 0.02) 10⁶ (0.82 ± 0.36) 10⁶ (1.36 ± 0.28) 10⁶Positive control: Lipofectin ® = (1.4 ± 0.2) 10⁸.

Assuming that the pH-sensitive liposomes deliver their content into thecytoplasm of the host cell, the naked plasmid must be able to penetratethe nucleus.

Assuming, hypothetically that the peptide-bis-acridines conjugates donot protect DNA from degradation, the observed transfection enhancementmust be the result of increased nuclear entry. The 4-5 fold increase oftransfection agrees with published results (Kaneda et al., supra Science(1989) 243:375-378) using proteins that bind to DNA and enhance DNAentry into the nucleus. Both the SNL peptide and WTcys peptide increaseexpression and are a convenient technique to target DNA into thenucleus.

EXAMPLE 8 Synthesis of Cationic Bile Salts

A. Preparation of the α-cholic Acid Amide of Nα-benyzlesterNε-tBOC-Lysine

The synthesis is based upon that of S. Bergstrom et al., Acta Chem Scand(1953) 7:1126. 204 mg (0.500 millimoles) of cholic acid was weighed intoa screw-capped test-tube, and 2.5 ml dioxane and 70 microliters (0.500millimoles) of triethylamine was added to the tube. The mixture wascooled in an ice bath until the solution solidified (at about 12° C). 65microliters (0.500 millimoles) of isobutyl chloroformate were added, thereaction tube was agitated and returned to the ice bath. The tube wasalternatively removed and replaced in the bath to keep the temperatureat the point of incipient solidification for 30 minutes.

Nα-benyzlester, Nε-tBOC-Lysine (0.500 millimoles) and 70 microliters(0.500 millimoles) of triethylamine were suspended in 0.6 ml of water.The mixture was cooled in the ice bath, added to the dioxane reactionmixture, and the container rinsed into the reaction mixture with another0.5 ml of ice water. The tube stood in the ice bath for ½ hour and thenwas permitted to warm to room temperature.

Most of the organic solvent was evaporated beneath a stream of argongas, and the residue was brought up to 3 ml with water. 5% aqueoussodium carbonate was added in a dropwise fashion until the pH reached 9.The mixture was extracted with three successive 3 ml portions of ethylether, and the aqueous phase saved.

To the aqueous residue, 0.5 N hydrochloric acid was added until the pHfell to 4. The mixture was extracted with three successive 3 ml portionsof ethyl ether, and the aqueous phase saved.

The pH of the aqueous residue was readjusted to 4 with 0.5 Nhydrochloric acid and extracted into five successive 3 ml portions ofethylacetate. These ethylacetate extracts were combined and evaporatedto dryness under vacuum to obtain 284 mg of colorless powder melting.The tBOC protecting group for the ε-amine was removed by standardmethods to yield the positively charged lysine derivative of cholicacid. In a similar fashion other positively charged derivatives ofcholic acid can be prepared.

B. Preparation of Cholic Acid Amide of Tris (2-aminoethyl)amine

When multiple amine groups are available for coupling to the activatedcholic acid the amine is added in a 6 fold excess over the activatedbile salt prepared as described in Example 8-A. The synthesis is basedupon that of Bergstrom et al., supra. Weigh 204 mg (0.500 millimoles) ofcholic acid into a screw-capped test tube. Add 2.5 ml dioxane and 70microliters (0.500 millimoles) of triethyl amine. Cool in an ice bathuntil the solution commences to solidify (at about 12° C.). Add 65microliters (0.500 millimoles) of isobutyl chloroformate and afteragitating, return the reaction tube to the ice bath. By alternatelyremoving from the ice bath and replacing in the bath, keep thetemperature at the point of incipient solidification for 30 minutes.

Add (3.00 millimoles) of tris(2-aminoethyl)amine and 70 microliters(0.500 millimoles) of triethylamine in 0.6 ml of water. Cool in the icebath, add to the dioxane reaction mixture, and rinse the container intothe reaction mixture with another 0.5 ml of ice water. Let stand in theice bath for ½ hour and then permit to warm to room temperature.

Evaporate most of the organic solvent beneath a stream of argon gas.Make the residue back up to 3 ml with water. Add 5% aqueous sodiumcarbonate dropwise until the pH reaches 7. Extract with three successive3 ml portions of ethyl ether, saving the aqueous phase.

To the aqueous residue add 0.5 N hydrochloric acid until the pH falls to4. Extract with three successive 3 ml portions of ethyl ether, savingthe aqueous phase.

Readjust the pH of the aqueous residue to 4 with 0.5 N hydrochloric acidand extract into five successive 3 ml portions of ethylacetate. Combinethese ethyl acetate extracts and evaporate to dryness under vacuum toobtain the cholic acid amide of tris(aminoethyl)amine.

EXAMPLE 9 Synthesis of Polyethyleneglycol-bis-Acridine

The synthesis of PEG-coupled bis-acridine spermidine follows standardchemistry and involves the following steps:

There are many methods for preparing activated monomethoxy PEG moleculesor activated PEG molecules. A preferred method has been described by D.Larwood and F. Szoka, J Labelled Comp & Radiopharm (1984) 21:603-614.Polyethylene glycol 1900 carbonyl-imidazole methyl ether was prepared bytaking 530 mg (0.28 mmol) dry PEG 1900 monomethyl ether in 2 ml drymethylene chloride and adding 78 mg (0.46 mmol) carbonyldiimidazole and10 mg (0.11 mmole) imidazole (sodium salt). After stirring overnight, 6ml dry methylene chloride were added and the mixture extracted with 3.75ml water, then dried with anhydrous sodium sulfate. After filtration,the solvent was removed, with quantitative yield. Alternatively, thesolvent was removed, and the resulting oil recrystallized fromchloroform/diethyl ether at −20° C. The resulting imidazole carbamatewhite crystals were filtered through a chilled funnel, rinsed with asmall amount of diethyl ether, and used immediately.

The imidazole carbamate (0.1 mM) is added to 0.125 mM ofN,N′-bis-(9-acridinyl)-4-aza-1,8-diaminooctane (“bis-acridinespermadine”, prepared as described by P.

Nielsen, Eur. J. Biochem. 122:283-289, 1992), dissolved in phenol andthe reaction run at 80° C. under argon for 2 hours. The mixture is takento dryness and the yellow product washed with cold ethanol and thendiethyl ether. The PEG is coupled via a carbamate linkage to thesecondary amine of the bis-acridine spermidine to form the monomethoxyPEG-bis-acridine spermidine and is soluble in water.

In a similar fashion the non-blocked PEG (molecular weight 6000), isactivated as above to form the bis-imidazole carbamate PEG. Thebis-imidazole carbamate PEG is reacted with a 2.5 fold excess ofbis-acridine spermidine to form the bis(bis-acridine spermidine)-PEG6000.

Various types of activators for PEG and monomethoxy PEG have beendescribed in U.S. Pat. No. 5,013,556 to Woodle et al. These methods canbe used to generate reactive PEGs that can be attached to thebis-acridine molecule via a variety of chemistries. For instance asulfhydryl containing monomethoxy-PEG can be attached to themaleimide-containing bis-acridine of Example 2-B.

EXAMPLE 10 DNA-Masking with PEG-bis-acridine

PEG molecules can be used to mask the surface of the DNA and permit theDNA to circulate for a longer period. Radio-iodinated plasmid DNA ismixed with monomethoxy-PEG-1900-bis-acridine spermidine as synthesizedin Example 9 at a 20 bp DNA-to-I PEG molecule ratio, for 30 minutes atroom temperature. An aliquot of the complex, 5 μg DNA in 0.2 ml PBS, isinjected via the tail vein into each of a group of 12 mice. Mice aresacrificed at various periods after injection. The blood and otherorgans are removed and the radioactivity associated with each organ isdetermined. DNA which has not been complexed to themonomethoxy-PEG-bis-acridine-spermidine is injected into a second groupof mice (control mice). After 10 minutes, 15% of the radioactive plasmidDNA remains in the blood in the control mice, whereas in the monomethoxyPEG-bis-acridine spermidine DNA group significantly greater levels ofthe radiolabeled plasmid-PEG complex remain in circulation. Thisindicates a pronounced masking effect of the DNA molecule by thePEG-bis-acridine spermidine.

EXAMPLE 11 Synthesis of a Lecithin Acyl Amine Masking Reagent

The synthesis of polynucleotide masking lipids is accomplished bystandard chemistry such as that described in C. Pidgeon et al., AnalBiochem (1989) 176:36-47.

The final reaction of the amine reactant with the lecithin imidazolideis undertaken immediately after formation of the lecithin imidazolide.The lecithin imidazolide (0.1 mM) is added to a solution of the amine(0.7 mM) in chloroform. Suitable amines for this coupling are listed inthis specification.

After two hours at room temperature the reaction mixture is added to atwo-fold volume of water/methanol and the pH is adjusted to 10. Thelecithin linked amine is extracted into the organic phase. The organicphase is then washed with 0.1 M sodium chloride and the organic phasetaken to dryness. The resulting acyl amine lecithin is used to mask thesurface of the polynucleotide. Various lysolecithin molecules can beused to prepare the lecithin-COOH, including lauroyl, myristoyl,palmitoyl, oleoyl or phytanoyl, or stearoyl. Other headgroups such asethanolamine or phosphatidic acid can be substituted for lecithin ifthey are suitably protected in the activation steps and deprotected atthe end of the reaction.

EXAMPLE 12 DNA-Masking with Lecithin Acyl Amine

The lecithin acyl amine of Example 11 can be added to DNA from anethanol solution at a ratio of 1 positive charge to each phosphate groupon the DNA. The molecule can be used to mask the surface of the DNA andpermit the DNA to circulate for a longer period. An aliquot of thecomplex, 5 μg DNA in 0.2 ml PBS, is injected via the tail vein into eachof a group of 12 mice. Mice are sacrificed at various periods afterinjection. The blood and other organs are removed and the radioactivityassociated with each organ is determined. DNA which has not beencomplexed to the lecithin acyl amine is injected into a second group ofmice (control mice). After 10 minutes, 15% of the radioactive plasmidDNA remains in the blood in the control mice, whereas in the lecithinacylamine group significantly greater levels of the radiolabeledplasmid-lecithin complex remain in. This indicates a pronounced maskingeffect of the DNA molecule by the lecithin acyl amine.

10 7 amino acids amino acid single linear not provided 1 Pro Lys Lys LysArg Lys Val 1 5 4 amino acids amino acid single linear not providedModified-site /note= “This position is N-formyl-.” 2 Xaa Met Leu Phe 1 8amino acids amino acid single linear not provided 3 Cys Ser Gly Arg GluAsp Val Trp 1 5 10 amino acids amino acid single linear not provided 4Ala Ala Phe Glu Asp Leu Arg Val Leu Ser 1 5 10 5 amino acids amino acidsingle linear not provided 5 Lys Arg Pro Arg Pro 1 5 5 amino acids aminoacid single linear not provided 6 Lys Phe Glu Arg Gln 1 5 16 amino acidsamino acid single linear not provided 7 Met Leu Ser Leu Arg Gln Ser IleArg Phe Phe Lys Pro Ala Thr Arg 1 5 10 15 13 amino acids amino acidsingle linear not provided 8 Cys Gly Tyr Gly Pro Lys Lys Lys Arg Lys ValGly Gly 1 5 10 13 amino acids amino acid single linear not provided 9Cys Gly Tyr Lys Pro Lys Val Arg Gly Lys Gly Lys Gly 1 5 10 30 aminoacids amino acid single linear not provided 10 Trp Glu Ala Ala Leu AlaGlu Ala Leu Ala Glu Ala Leu Ala Glu 1 5 10 15 His Leu Ala Glu Ala LeuAla Glu Ala Leu Glu Ala Leu Ala Ala 16 20 25 30

We claim:
 1. A composition for presenting a polynucleotide to asubcellular component of a eukaryotic cell comprising: a) apolynucleotide; and b) three or more functional agents associated withthe polynucleotide, the agents being selected from the group consistingof: i) a cell recognition agent capable of recognizing the eukaryoticcell, said agent comprising a ligand for a receptor located on theeukaryotic cell surface and a DNA-associating moiety being coupledthereto, the DNA-associating moiety being selected from the groupconsisting of a single stranded polynucleotide linker, a dendrimerpolycation, a major-or minor groove binder, and an intercalating agent;ii) a membrane-permeabilizing agent capable of transporting thepolynucleotide across the cytoplasmic membrane of the eukaryotic cell;iii) a subcellular-localization agent capable of delivering thepolynucleotide from the cytoplasm of the eukaryotic cell to asubcellular component of the eukaryotic cell, said agent furthercomprising a DNA-associating moiety being coupled thereto, theDNA-associating moiety being selected from the group consisting of asingle stranded polynucleotide linker, a dendrimer polycation, amajor-or minor groove binder, and an intercalating agent; and iv) apolynucleotide-masking agent capable of increasing the circulatoryhalf-life of the desired polynucleotide.
 2. A composition for presentinga polynucleotide to a subcellular component of a eukaryotic cellcomprising: a) a polynucleotide; b) an agent operatively coupled to thepolynucleotide, the agent being selected from the group consisting of i)a cell recognition agent capable of recognizing the eukaryotic cell,said agent comprising a ligand for a receptor located on the eukaryoticcell surface and a DNA-associating moiety being coupled thereto, theDNA-associating moiety being selected from the group consisting of asingle stranded polynucleotide linker, a dendrimer polycation, amajor-or minor-groove binder, and an intercalating agent; ii) amembrane-permeabilizing agent capable of transporting the polynucleotideacross the cytoplasmic membrane of the eukaryotic cell; and iii) asubcellular-localization agent capable of delivering the polynucleotidefrom the cytoplasm of the eukaryotic cell to a subcellular component ofthe eukaryotic cell; and c) a DNA masking agent capable of increasingthe circulatory half-life of the polynucleotide, wherein the DNA-maskingagent has the chemical formula

wherein n is an integer of 6 to 24; Y is selected from the groupconsisting of hydroxy, ethanolamine, choline, glycerol, serine andinositol; R¹ is (C₆-C₂₄) is alkyl or (C₆-C₂₄) alkenyl; R³ is H, or(C₁-C₁₀) alkyl or (C₁-C₁₀) alkylamine; and R⁴ is a positively chargedlinear or branched (C₁-C₃₀) alkyl or (C₁-C₃₀) alkylamine, wherein one ormore of the carbon atoms may be substituted with NR′, wherein R′ is H or(C₁-C₁₀) alkyl or (C₁-C₁₀) alkylamine.
 3. A composition for presenting apolynucleotide to a subcellular component of a eukaryotic cellcomprising a) a polynucleotide; b) an agent operatively coupled to thepolynucleotide, the agent being selected from the group consisting of i)a cell recognition agent capable of recognizing the eukaryotic cell,said agent comprising a ligand for a receptor located on the eukaryoticcell surface and a DNA-associating moiety being coupled thereto, theDNA-associating moiety being selected from the group consisting of asingle stranded polynucleotide linker, a dendrimer polycation, amajor-or minor groove binder, and an intercalating agent; ii) amembrane-permeabilizing agent capable of transporting the polynucleotideacross the cytoplasmic membrane of the eukaryotic cell; and iii) asubcellular-localization agent capable of delivering the polynucleotidefrom the cytoplasm of the eukaryotic cell to a subcellular component ofthe eukaryotic cell; and c) a DNA masking agent capable of increasingthe circulatory half-life of the polynucleotide, wherein the maskingagent comprises polyethylene glycol (PEG) covalently linked to aDNA-associating moiety.
 4. A composition for presenting a polynucleotideto a subcellular component of a eukaryotic cell comprising apolynucleotide; a membrane-permeabilizing agent capable of transportingthe polynucleotide across the cytoplasmic membrane of the eukaryoticcell operatively coupled to the polynucleotide, wherein themembrane-permeabilizing agent comprises an amphipathic peptide; and alipid or a polyamine.
 5. The composition of claim 4, wherein theamphipathic peptide comprises GALA/SEQ ID NO:
 10. 6. The composition ofclaim 4, wherein the peptide comprises a cyclic peptide.
 7. Thecomposition of claim 6, wherein the cyclic peptide is selected from thegroup consisting of gramicidin S and tyrocidines.
 8. The composition ofclaim 7, wherein the cyclic peptide comprises gramicidin S.
 9. Acomposition for presenting a polynucleotide to a subcellular componentof a eukaryotic cell comprising a polynucleotide; and a cell recognitionagent operatively coupled to the polynucleotide and capable ofrecognizing the eukaryotic cell, said agent comprising a ligand for areceptor located on the eukaryotic cell surface and a DNA-associatingmoiety being coupled thereto, the DNA-associating moiety comprising anintercalating agent, wherein the intercalating agent has the formula

wherein Z comprises a bond, a reactive group selected from the groupconsisting of N-hydroxysuccinimide, maleimide, maleimidophenyl, pyridyldisulfide, hydrazide, and phenylglyoxal, or ZY, wherein Y is selectedfrom the group consisting of cell surface receptor ligands, nuclearlocalization sequences, and membrane permeabilizing components; n and mare independently an integer of 1 to 20; p is an integer of 0 to 20; andAr₁ and Ar₂ are independently selected from the group consisting ofethidium bromide, acridine, mitoxanotrone, oxazolopyridocarbazole,ellipticine, N-methyl-2, 7-diazapyrenium, and derivatives capable ofintercalating a polynucleotide.
 10. The composition of claim 9, whereinthe intercalating agent is coupled to a plurality of ligands.
 11. Acomposition for presenting a polynucleotide to a subcellular componentof a eukaryotic cell comprising a polynucleotide; and a cell recognitionagent operatively coupled to the polynucleotide and capable ofrecognizing the eukaryotic cell, said agent comprising a ligand for areceptor located on the eukaryotic cell surface and a DNA-associatingmoiety being coupled thereto, the DNA-associating moiety comprising anintercalating agent, wherein the intercalating agent comprises thetrigalactosylated spermidine bis-acridine compound (26) of FIG.
 13. 12.A composition for presenting a polynucleotide to a subcellular componentof a eukaryotic cell comprising a polynucleotide; asubcellular-localization agent operatively coupled to the polynucleotideand capable of delivering the polynucleotide from the cytoplasm of theeukaryotic cell to a subcellular component of the eukaryotic cellcomprising a nuclear localization segment coupled to a DNA-associatingmoiety, wherein the DNA-associating moiety comprises an intercalatingagent having the chemical formula

wherein comprises a bond, a reactive group selected from the groupconsisting of N-hydroxysuccinimide, maleimide, maleimidophenyl, pyridyldisulfide, hydrazide, and phenylglyoxal, or ZY, wherein Y is selectedfrom the group consisting of cell surface receptor ligands, nuclearlocalization sequences, and membrane permeabilizing components; n and mare independently an integer of 1 to 20; p is an integer of 0 to 20; andAr₁ and Ar₂ are independently selected from the group consisting ofetuidium bromide, acridine, mitoxantrone, oxazolopyridocarbazole,ellipticine, N-methyl-2, 7-diazapyrenium, and derivatives capable ofintercalating a polynucleotide.
 13. The composition of claim 12, whereinAr₁ and Ar₂ comprise acridine.
 14. The composition of claim 13, whereinZ comprises the reactive group maleimidophenyl.
 15. A composition forpresenting a polynucleotide to a subcellular component of a eukaryoticcell comprising a polynucleotide; a subcellular-localization agentoperatively coupled to the polynucleotide and capable of delivering thepolynucleotide from the cytoplasm of the eukaryotic cell to asubcellular component of the eukaryotic cell comprising a nuclearlocalization segment coupled to a DNA-associating moiety, wherein theDNA-associating moiety comprises an intercalating agent having thechemical formula


16. The composition of claim 12, wherein Z comprises the group


17. A composition for presenting a desired polynucloetide to asubcellular component of a eukaryotic cell comprising: a) a desiredpolynucleotide operatively coupled to a polynucleotide-associatingmoiety selected from the group consisting of: i) an intercalator; ii) alinker strand comprising a single stranded polynucleotide; iii) adendrimer polycation; and b) three functional agents operatively coupledto the polynucleotide, the function agents selected from the groupconsisting of: i) a cell recognition agent capable of recognizing theeukaryotic cell; ii) membrane-permeabilizing agent capable oftransporting the desired polynucleotide across the cytoplasmic membraneof the eukaryotic cell; iii) a subcellular-localization agent capable ofdelivering the desired polynucleotide from the cytoplasm of theeukaryotic cell to a subcellular component of the eukaryotic cell; iv) apolynucleotide-masking agent capable of increasing the circulatoryhalf-life of the desired polynucleotide; and v) a group to link with oneof more of the functional agents i)-iv) by a covalent bond.
 18. Thecomposition of claim 17 wherein the polynucleotide-associating moietycomprises an intercalator.
 19. The composition of claim 18 wherein theintercalator is coupled to a plurality of the functional agents i)-iv).20. The composition of claim 18 wherein the intercalator has the formula

wherein Z comprises a reactive group selected from the group consistingof N-hydroxysuccinimide, maleimide, maleimidophenyl, pyridyl disulfide,hydrazide, and phenylglyoxal, or ZY, wherein Y is selected from thegroup consisting of functional agents i)-iv); n and m are independentlyan integer of 1 to 20; p is an integer of 0 to 20; and Ar₁ and Ar₂ areindependently selected from the group consisting of ethidium bromide,acridine, mitoxanotrone, oxazolopyridocarbazole, ellipticine,N-methyl-2, 7-diazapyrenium, and derivatives capable of intercalating apolynucleotide.
 21. The composition of claim 20 wherein Ar₁ and Ar₂ eachcomprise acridine.
 22. The composition of claim 21 wherein theintercalator comprises the trigalactosylated spermidine bis-acridinecompound (26) of FIG.
 13. 23. The composition of claim 20 wherein Zcomprises the group maleimidophenyl.
 24. The composition of claim 20wherein Z comprises the group


25. The composition of claim 18 wherein the intercalator has thechemical formula


26. The composition of claim 18 wherein the intercalator isbiodegradable.
 27. The composition of claim 26 wherein the intercalatorcomprises a peptide sequence and intercalating groups linked to thepeptide sequence.
 28. The composition of claim 27 wherein the peptidesequence comprises KK.
 29. The composition of claim 18 wherein theintercalator has the formula

wherein Ar₁ and Ar₂ are independently selected from the group consistingof ethidium bromide, acridine, mitoxanotrone, oxazolopyridocarbazole,ellipticine, N-methyl-2, 7-diazapyrenium, and derivatives capable ofintercalating a polynucleotide; each aa is independently an amino acid;x and z are integers independently selected from 1 to 100; y is aninteger from 0 to 5; aa₁ and aa₂ are lysine residues; and N¹ and N² arenitrogens from the ε-amino groups of the lysine residues aa₁ and aa₂.30. The composition of claim 17 wherein the polynucleotide-associatingmoiety comprises a linker strand complementary to the desiredpolynucleotide.
 31. The composition of claim 30 wherein substantiallythe entire linker strand sequence is complementary to the desiredpolynucleotide.
 32. The composition of claim 30 wherein the linkerstrand comprises an extension of the desired polynucleotide.
 33. Thecomposition of claim 30 wherein the linker strand has a sequence with aplurality of regions, each region complementary to a portion of thedesired polynucleotide.
 34. The composition of claim 30 wherein thepolynucleotide-associating moiety comprises a plurality of linkerstrands.
 35. The composition of claim 30 wherein thepolynucleotide-associating moiety comprises a first linker complementaryto the desired polynucleotide and a second linker complementary to thedesired polynucleotide.
 36. The composition of claim 17 wherein thepolynucleotide-associating moiety comprises a dendrimer polycation. 37.The composition of claim 17 wherein the polynucleotide-associatingmoiety comprises a major- or minor-groove binder.
 38. The composition ofclaim 37 wherein the major- or minor-groove binder is selected from thegroup consisting of distamycin A and Hoechst dye
 33258. 39. Thecomposition of claim 17 wherein the functional agent comprises a cellrecognition agent.
 40. The composition of claim 17 wherein thefunctional agent comprises a membrane-permeabilization agent.
 41. Thecomposition of claim 17 further comprising a membrane-permeabilizerselected from the group consisting of polylysine, polyarginine, poly(lysine-arginine), polyamines, dendrimer polycations, cationic bilesalts and amphipathic peptides.
 42. The composition of claim 17 whereinthe functional agent comprises a subcellular-localization agent selectedfrom the group consisting of a nuclear-localization component, alysosomal-localization component and a mitochondrial-localizationcomponent.
 43. The composition of claim 42 wherein thesubcellular-localization agent is selected from the group consisting ofthe sequence PKKKRKV/SEQ ID NO. 1, the sequence AAFEDLRVLS/SEQ ID NO. 4,the sequence KRPRP/SEQ ID NO. 5, the sequence KFERQ/SEQ ID NO. 6 and thesequence MLSLRQSIRFFKPATR/SEQ ID NO
 7. 44. The composition of claim 17wherein the functional agent comprises a polynucleotide-masking agent.45. The composition of claim 44 wherein the polynucleotide-masking agentcomprises polyethylene glycol linked to the polynucleotide-associatingmoiety.
 46. The composition of claim 45 comprising polyethyleneglycol-bis-acridine.
 47. A composition for presenting a desiredpolynucleotide to a subcellular component of a eukaryotic cellcomprising a desired polynucleotide associated with a cationic bile salthaving the formula

wherein X and Y are independently H or OH; R³ is hydrogen (C₁-C₁₀) alkylor (C₁-C₁₀) alkylamine; and R⁴ is a positively charged linear orbranched (C₁-C₃₀) alkyl or (C₁-C₃₀) alkylamine, wherein one or more ofthe carbon atoms may be substituted with NR′, wherein R′ is H,(C₁-C₁₀)alkyl or (C₁-C₁₀) alkylamine.
 48. The composition of claim 47further comprising a lipid.
 49. A composition for presenting a desiredpolynucleotide to a subcellular component of a eukaryotic cellcomprising a desired polynucleotide associated with apolynucleotide-masking agent having the chemical formula

wherein n is an integer of 6 to 24; Y is selected from the groupconsisting of hydroxy, ethanolamine, choline, glycerol, serine andinositol; R¹ is (C₆-C₂₄) alkyl or (C₆-C₂₄) alkenyl; R³ is H, or (C₁-C₁₀)alkyl or (C₁-C₁₀) alkylamine; and R⁴ is a positively charged linear orbranched (C₁-C₃₀) alkyl or (C₁-C₃₀) alkylamine, wherein one or more ofthe carbon atoms may be substituted with NR′, wherein R′ is H or(C₁-C₁₀) alkyl or (C₁-C₁₀) alkylamine.
 50. The composition of claim 49wherein the lipid comprises lecithin acyl amine.
 51. A composition forpresenting a desired polynucleotide to a subcellular component of aeukaryotic cell comprising a desired polynucleotide associated with anamphipathic peptide capable of assuming a pH dependent α-helixconformation.
 52. The composition of claim 51 further comprising alipid.
 53. The composition of claim 51 further comprising a polyamine.54. The composition of 4 wherein the amphipathic peptide comprises anamphipathic peptide capable of assuming a β-pleated sheet conformation.55. The composition of 54 wherein the β-pleated sheet amphipathicpeptide has a first and second face such that the first face ispositively charged and the second face is substantially neutral.
 56. Thecomposition of claim 4 wherein the amphipathic peptide comprises acyclic peptide.
 57. The composition of claim 56 wherein the cyclicpeptide is selected from the group consisting of tyrocidines andgramicidin S.
 58. The composition of claim 56 wherein the cyclic peptidecomprises gramicidin S.
 59. The composition of claim 4 comprisinggramicidin S and dioleoylphosphatidylethanolamine.
 60. The compositionof claim 59 comprising gramicidin S and dioleoylphosphatidylethanolaminein a molar ratio of greater than about 1:1.
 61. The composition of claim60 comprising gramicidin S and dioleoylphosphatidylethanolamine in amolar ratio of greater than about 5:1.
 62. The composition of claim 51wherein the amphipathic peptide α-helix comprises a first and a secondaxial face such that the first face is substantially charged and thesecond face is substantially neutral.
 63. The composition of claim 62wherein the first face is negatively charged.
 64. The composition ofclaim 63 wherein the amphipathic peptide comprises the sequence GALA/SEQID NO.
 10. 65. The composition of claim 1 wherein one or more of thefunctional agents is operatively coupled to the desired polynucleotideby a polynucleotide-associating moiety.
 66. The composition of claim 65wherein the polynucleotide-associating moiety is selected from the groupconsisting of a single stranded polynucleotide linker, a dendrimerpolycation, a major- or minor-groove binder, and an intercalator. 67.The composition of claim 47 further comprisingdioleoylphosphatidylethanolamine.